Plant protein disulfide isomerase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a protein disulfide isomerase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the protein disulfide isomerase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the protein disulfide isomerase in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/049,408, filed Oct. 15, 1998.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingprotein disulfide isomerases in plants and seeds.

BACKGROUND OF THE INVENTION

Protein folding requires the assistance of folding helpers in vivo. Theformation or isomerization of disulfide bonds in proteins is a slowprocess requiring catalysis. In nascent polypeptide chains the cysteineresidues are in the thiol form. The formation of the disulfide bondsusually occurs simultaneously with the folding of the polypeptide, inthe endoplasmic reticulum of eukaryotes or in the periplasm ofGram-negative bacteria. Cells contain three types of accessory proteinsthat function to assist polypeptides in folding to their nativeconformations: protein disulfide isomerases, propyl cis-transisomerases, and molecular chaperones.

Protein disulfide isomerase (PDI) is a homodimeric eukaryotic enzymewhich catalyzes disulfide interchange reactions. PDI is also thought tobe the beta subunit of the heterotetramer prolyl hydrolase, the enzymethat hydroxylates the proline residues in Collagen. PDI appears tobelong to a family of closely related proteins which have specificfunctions. PDI (EC 5.3.4.1), also called S-S rearrangase, catalyzes therearrangement of both intrachain and interchain disulfide bonds inproteins to form native structures. The reaction depends onsulfhydryl-disulfide interchange, and PDI needs reducing agents orpartly-reduced enzyme. A family of PDI-like proteins has been identifiedin mammals, yeasts, fungi, plants, and Drosophila.

In Drosophila, a PDI precursor was identified by screening a genomic DNAlibrary at reduced stringency hybridization conditions using a ratPhospholipase C alpha cDNA probe. Northern analysis showed that thisgene encodes a transcript that is present throughout development, inheads and bodies of adults. The encoded protein contains two domainsexhibiting high similarity to thioredoxin, two regions that are similarto the hormone binding domain of human estrogen receptor, and aC-terminal ER-retention signal (KDEL). Overall, this Drosophila PDI genecontains a higher similarity to rat protein disulfide isomerase (53%identical) than to rat Phospholipase C alpha (30% identical) (McKay etal. (1995) Insect Biochem. Mol. Biol. 25:647-654).

Another member of the PDI family is ERp60, a PDI isoform initiallymisidentified as a phosphatidylinositol-specific phospholipase C. Thehuman and Drosophila ERp60 polypeptides have been cloned and expressed.These two ERp60 polypeptides are similar to human PDI within almost alltheir domains, the only exception being the extreme C-terminal region.Coexpression in insect cells of the human or Drosophila ERp60 with thealpha subunit of human propyl 4-hydrolase does not result in tetramerformation or prolyl 4-hydroxylase activity in the cells. This lack oftetramer formation is not only due to the differences in the C-terminalregion since no prolyl 4-hydroxylase tetramer is formed when a humanERp60 hybrid containing the C-terminal region of the human PDIpolypeptide is used (Koivunen et al. (1996) Biochem. J. 316:599-605).The 5′ flanking region of the ERp60 gene has no TATAA box or CCAAT motifbut contains several potential binding sites for transcription factors.The highest levels of expression of the human ERp60 mRNA are found inthe liver, placenta, lung, pancreas, and kidney, and the lowest in theheart, skeletal muscle, and brain. The ERp60 gene has been mapped byfluorescence in situ hybridization to 15q15, a different chromosome thanwhere the human PDI and thioredoxin genes are found (Koivunen et al.(1997) Genomics 42:397-404).

Full-length cDNA clones encoding two members of the mice PDI family havebeen cloned, sequenced, and expressed (ERp59/PDI and ERp72). ERp59/PDIhas been identified as the microsomal PDI. The ERp72 amino acid sequenceshares sequence identity with ERp59/PDI at three discrete regions,having three copies of the sequences that are thought to be theCGHC-containing active sites of ERp59/PDI. ERp59/PDI has the sequenceLys-Asp-Glu-Leu at its COOH terminus while ERp72 has the relatedsequence Lys-Glu-Glu-Leu (Mazzarella et al. (1990) J. Biol. Chem.265:1094-1101). A cDNA clone containing sequence similarity to themammalian lumenal endoplasmic reticulum protein ERp72 has been isolatedfrom an alfalfa (Medicago sativa L.) cDNA library by screening with acDNA encoding human PDI. The polypeptide encoded by this cDNA possessesa putative N-terminal secretory signal sequence and two regionsidentical to the active sites of PDI and ERp72. This protein appears tobe encoded by a small gene family in alfalfa, whose transcripts areconstitutively expressed in all major organs of the plant. In alfalfacell suspension cultures, ERp72 transcripts are induced by treatmentwith tunicamycin, but not in response to calcium ionophore, heat shockor fungal elicitor (Shorrosh and Dixon (1992) Plant J. 2:51-58).

Another member of the PDI family is ERp5. The amino acid sequencededuced from this cDNA insert contains two copies of the 11-amino-acidsequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copiesof this sequence are found in the active sites of rat and human PDI andin Form I phosphoinositide-specific phospholipase C. Genomic sequencessimilar to the cDNA clone are amplified 10-20-fold in hamster cellsselected for resistance to increasing concentrations of hydroxyurea, aphenomenon observed earlier with cDNA clones for the M2 subunit ofribonucleotide reductase and ornithine decarboxylase. RNA blots probedwith ERp5 cDNA show two poly(A)+ RNA species which are elevated inhydroxyurea-resistant cells (Chaudhuri et. al. (1992) Biochem. J.281:645-650).

A PDI-like protein from Acanthamoeba castellanii contains two highlyconserved thioredeoxin-like domains, each about 100 amino acids.However, the A. castellanii PDI-like protein differs from other membersin many aspects, including the overall organization and isoelectricpoint. Southern and Northern analyses demonstrate that the PDI-likeprotein is encoded by a single-copy gene which is transcribed togenerate a 1500-nucleotide mRNA (Wong and Bateman (1994) Gene150:175-179).

The Chlamydomonas RB60 gene encodes a chloroplast-localized PDI which isinvolved in the redox-regulated binding of chloroplast poly(A)-bindingprotein to the 5′-leader region of psbA mRNA. Protein disulfideisomerase RB60 regulates chloroplast translational activation (Kim andMayfield (1997) Science 278:1954-1957).

High level gene expression does not always lead to corresponding highlevel secretion of heterologous proteins. The rate limiting step hasbeen shown, in many cases, to be the processing and exit of the proteinfrom the endoplasmic reticulum. Proteins or peptides with high levels ofdisulfide bonds can be adversely affected during expression. Therefore,coexpression and/or overexpression of PDIs could significantly enhanceexpression levels of many heterologous proteins. An example would be thecoexpression of PDIs with insect-selective neurotoxins, since many ofthese are highly enriched in cysteines and feature multiple disulfidebonds.

Protein disulfide isomerases have been described in alfalfa (2 genes andone probable PDI P5 homolog), barley (2 genes, and one probable PDI P5homolog), maize, wheat, tobacco, and castor bean. In addition, based onsequence similarity to other known PDIs, two putative protein disulfideisomerases have been identified in Arabidopsis. Included in thisapplication are corn, and soybean ESTs with sequence similarities toprotein disulfide isomerase precursor. The corn sequences included shareno similarity with the known maize PDI. Also included are corn, balsampear, soybean, and the wheat ESTs with sequence similarities to RB60.Presently there are no plant RB60-homologs in the public domain.Overexpression of any of these PDIs together with another foreignprotein will result in an increased yield of secreted, active foreignprotein due to proper folding of the foreign protein.

Present in the NCBI database are corn and soybean sequences withsimilarities to the polynucleotides included in the present application.These ESTs have NCBI General Identifier NOs:4289796, 4827500, 5124153,5325044, 5361231, 5525515, 5597319, 5650368, 5688597, 5714111, 5770161,and 5804735.

SUMMARY OF THE INVENTION

The present invention relates to isolated polynucleotides comprising anucleotide sequence encoding a protein disulfide isomerase precursorpolypeptide of at least 100 amino acids that has at least 85% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of a corn protein disulfide isomeraseprecursor polypeptide selected from the group consisting of SEQ ID NO:2,and SEQ ID NO:6, a soybean protein disulfide isomerase precursorpolypeptide of SEQ ID NO:4. The present invention relates to isolatedpolynucleotides comprising a nucleotide sequence encoding an RB60polypeptide of at least 100 amino acids that has at least 85% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of a balsam pear RB60 polypeptide ofSEQ ID NO:8, a corn RB60 polypeptide selected from the group consistingof SEQ ID NO:10 and SEQ ID NO:12, a soybean RB60 polypeptide selectedfrom the group consisting of SEQ ID NO:14 and SEQ ID NO:16, and a wheatRB60 polypeptide selected from the group consisting of SEQ ID NO:18 andSEQ ID NO:20. The present invention also relates to an isolatedpolynucleotide comprising the complement of the nucleotide sequencesdescribed above.

It is preferred that the isolated polynucleotides of the claimedinvention consist of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 thatcodes for the polypeptide selected from the group consisting of SEQ IDNOs:2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. The present invention alsorelates to an isolated polynucleotide comprising a nucleotide sequencesof at least one of 40 (preferably at least one of 30, most preferably atleast one of 15) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15, 17, and 19 and the complement of such nucleotide sequences.

The present invention relates to a chimeric gene comprising an isolatedpolynucleotide of the present invention operably linked to suitableregulatory sequences.

The present invention relates to an isolated host cell comprising achimeric gene of the present invention or an isolated polynucleotide ofthe present invention. The host cell may be eukaryotic, such as a yeastor a plant cell, or prokaryotic, such as a bacterial cell or virus. Avirus host cell of the present invention is preferably a baculovirus.The baculovirus preferably comprises an isolated polynucleotide of thepresent invention or a chimeric gene of the present invention.

The present invention relates to a process for producing an isolatedhost cell comprising a chimeric gene of the present invention or anisolated polynucleotide of the present invention, the process comprisingeither transforming or transfecting an isolated compatible host cellwith a chimeric gene or isolated polynucleotide of the presentinvention.

The present invention relates to a protein disulfide isomerase precursoror an RB60 polypeptide of at least 100 amino acids comprising at least85% homology based on the Clustal method of alignment compared to apolypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, 16, 18, and 20.

The present invention relates to a method of selecting an isolatedpolynucleotide that affects the level of expression of a proteindisulfide isomerase precursor or an RB60 polypeptide in a host cell, themethod comprising the steps of:

-   -   constructing an isolated polynucleotide of the present invention        or an isolated chimeric gene of the present invention;    -   introducing the isolated polynucleotide or the isolated chimeric        gene into a host cell (preferably a plant cell);    -   measuring the level a protein disulfide isomerase precursor or        an RB60 polypeptide in the plant cell containing the isolated        polynucleotide; and    -   comparing the level of a protein disulfide isomerase precursor        or an RB60 polypeptide in the host cell containing the isolated        polynucleotide with the level of a protein disulfide isomerase        precursor or an RB60 polypeptide in a host cell that does not        contain the isolated polynucleotide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a protein disulfide isomeraseprecursor or an RB60 polypeptide gene, preferably a plant proteindisulfide isomerase precursor or an RB60 polypeptide gene, comprisingthe steps of: synthesizing an oligonucleotide primer comprising anucleotide sequence of at least one of 40 (preferably at least one of30, most preferably at least one of 15) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and the complement of suchnucleotide sequences; and amplifying a nucleic acid fragment (preferablya cDNA inserted in a cloning vector) using the oligonucleotide primer.The amplified nucleic acid fragment preferably will encode a portion ofa protein disulfide isomerase precursor or an RB60 amino acid sequence.

The present invention also relates to a method of obtaining a nucleicacid fragment encoding all or a subsantial portion of the amino acidsequence encoding a protein disulfide isomerase precursor or an RB60polypeptide comprising the steps of: probing a cDNA or genomic librarywith an isolated polynucleotide of the present invention; identifying aDNA clone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying Sequence Listing which form a part ofthis application.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Protein Disulfide IsomerasesSEQ ID NO: Protein Clone Designation (Nucleotide) (Amino Acid) Corn PDIprecursor cr1n.pk0090.d2 1 2 Soybean PDI srr3c.pk002.a8 3 4 precursorCorn PDI precursor csi1.pk0032.c9 5 6 Balsam Pear RB60 fds.pk0022.c11 78 Corn PDI RB60 Contig of: 9 10 cen3n.pk0155.e7 cs1.pk0100.a7p0032.crcbb52r p0125.czabp07r Corn PDI RB60 cs1.pk0077.f10 11 12 SoybeanPDI RB60 sr1.pk0095.e9 13 14 Soybean PDI RB60 Contig of: 15 16scr1c.pk005.i17 sdp2c.pk038.e22 sdp3c.pk021.a3 sfl1.pk0026.h1sl2.pk0075.b10 Wheat PDI RB60 wl1n.pk0027.f4 17 18 Wheat PDI RB60wre1n.pk0015.d10 19 20

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “polynucleotide” is a nucleotide sequence such as anucleic acid fragment. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, or synthetic DNA. An isolated polynucleotide of the presentinvention may include at least one of 40 contiguous nucleotides,preferably at least one of 30 contiguous nucleotides, most preferablyone of at least 15 contiguous nucleotides, of the nucleic acid sequenceof the SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 40 (preferably at least one of30, most preferably at least one of 15) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and the complement of suchnucleotide sequences may be used in methods of selecting an isolatedpolynucleotide that affects the expression of a polypeptide (such as PDIprecursor or PDI RB60) in a host cell. A method of selecting an isolatedpolynucleotide that affects the level of expression of a polypeptide ina host cell (eukaryotic, such as plant, or prokarotic such as yeastbacterial or virus) may comprise the steps of: constructing an isolatedpolynucleotide of the present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or theisolated chimeric gene into a host cell; measuring the level apolypeptide in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide in the host cell containing theisolated polynucleotide with the level of a polypeptide in a host cellthat does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Haamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are 80% identical to theamino acid sequences reported herein. Preferred nucleic acid fragmentsencode amino acid sequences that are 85% identical to the amino acidsequences reported herein. More preferred nucleic acid fragments encodeamino acid sequences that are 90% identical to the amino acid sequencesreported herein. Most preferred are nucleic acid fragments that encodeamino acid sequences that are 95% identical to the amino acid sequencesreported herein. Suitable nucleic acid fragments not only have the aboveidentities but typically encode a polypeptide having at least 50 aminoacids, preferably 100 amino acids, more preferably 150 amino acids,still more preferably 200 amino acids, and most preferably 250 aminoacids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several PDIs havebeen isolated and identified by comparison of random plant cDNAsequences to public databases containing nucleotide and proteinsequences using the BLAST algorithms well known to those skilled in theart. The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding homologous proteins from the same orother plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other PDIs, either as cDNAs or genomic DNAs,could be isolated directly by using all or a portion of the instantnucleic acid fragments as DNA hybridization probes to screen librariesfrom any desired plant employing methodology well known to those skilledin the art. Specific oligonucleotide probes based upon the instantnucleic acid sequences can be designed and synthesized by methods knownin the art (Maniatis). Moreover, the entire sequences can be useddirectly to synthesize DNA probes by methods known to the skilledartisan such as random primer DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast one of 40 (preferably one of at least 30, most preferably one ofat least 15) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,15, 17, 19 and the complement of such nucleotide sequences may be usedin such methods to obtain a nucleic acid fragment encoding a substantialportion of an amino acid sequence of a polypeptide (such as PDIprecursor or PDI RB 60). The present invention relates to a method ofobtaining a nucleic acid fragment encoding a substantial portion of apolypeptide of a gene (such as PDI precursor or PDI RB 60) preferably asubstantial portion of a polypeptide of a plant gene, comprising thesteps of: synthesizing an oligonucleotide primer comprising a nucleotidesequence of at least one of 40 (preferably at least one of 30, mostpreferably at least one of 15) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19 and the complement of such nucleotidesequences; and amplifying a nucleic acid fragment (preferably a cDNAinserted in a cloning vector) using the oligonucleotide primer. Theamplified nucleic acid fragment preferably will encode a portion of apolypeptide (such as PDI precursor or PDI RB 60).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher levels than normal or in cell types or developmental stages inwhich they are not normally found. This would have the effect ofaltering the level of properly folded proteins in those cells.Coexpression of a member of the PDI family with another foreign proteinwill result in a greater yield of active, secreted foreign protein dueto the improvement in proper folding done by the PDI.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded protein disulfide isomerases. An example of a vector forhigh level expression of the instant polypeptides in a bacterial host isprovided (Example 7).

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries: Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various balsam pear, corn, rice,soybean and wheat tissues were prepared. The characteristics of thelibraries are described below. TABLE 2 cDNA Libraries from Balsam Pear,Corn, Rice, Soybean and Wheat Library Tissue Clone cen3n Corn Endosperm20 Days After cen3n.pk0155.e7 Pollination* cr1n Corn Root From 7 Day OldSeedlings* cr1n.pk0090.d2 cs1 Corn Leaf Sheath From 5 Week Old Plantcs1.pk0077.f10 cs1 Corn Leaf Sheath From 5 Week Old Plant cs1.pk0100.a7csi1 Corn Silk csi1.pk0032.c9 fds Momordica charantia Developing Seedfds.pk0022.c11 p0032 Corn Regenerating Callus p0032.crcbb52r (Hi-II 223aand 1129e), 10 and 14 Days After Auxin Removal, Pooled p0125 Corn AntherProphase I* p0125.czabp07r scr1c Soybean Embryogenic Suspension Culturescr1c.pk005.i17 Subjected to 4 Vacuum Cycles and Collected 12 HoursLater sdp2c Soybean Developing Pods (6-7 mm) sdp2c.pk038.e22 sdp3cSoybean Developing Pods (8-9 mm) sdp3c.pk021.a3 sfl1 Soybean ImmatureFlower sfl1.pk0026.h1 sl2 Soybean Two-Week-Old Developing sl2.pk0075.b10Seedlings Treated With 2.5 ppm chlorimuron sr1 Soybean Rootsr1.pk0095.e9 srr3c Soybean 8-Day-Old Root srr3c.pk002.a8 wl1n WheatLeaf From 7 Day Old Seedling* wl1n.pk0027.f4 wre1n Wheat Root From 7 DayOld wre1n.pk0015.d10 Etiolated Seedling**These libraries were normalized essentially as described in U.S. Pat.No. 5,482,845, incorporated herein by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding protein disulfide isomerases were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) Nat.Genet. 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Protein DisulfideIsomerase Precursor

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to proteindisulfide isomerase precursor from Humicola insolens or Bos taurus (NCBIGeneral Identifier Nos. 1709618 and 129726, respectively). Shown inTable 3 are the BLAST results for individual ESTs (“EST”), or thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”): TABLE 3 BLAST Results for Sequences EncodingPolypeptides Homologous to Protein Disulfide Isomerase Precursor NCBIGeneral BLAST Clone Status Identifier No. pLog Score cr1n.pk0090.d2 EST1709618 55.52 srr3c.pk002.a8 EST 1709618 48.52 csi1.pk0032.c9:fis FIS129726 28.04

The data in Table 4 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 4 and 6 and theHumicola insolens and Bos taurus sequences (NCBI General Identifier Nos.1709618 and 129726, respectively). TABLE 4 Percent Identity of AminoAcid Sequences Deduced From the Nucleotide Sequences of cDNA ClonesEncoding Polypeptides Homologous to Protein Disulfide IsomerasePrecursor Percent Identity to SEQ ID NO. 1709618 129726 2 80.0 42.7 417.0 22.2 6 50.0 37.3

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of two corn and one soybean proteindisulfide isomerase precursor. These sequences represent the first cornand soybean sequences encoding protein disulfide isomerase precursor.

Example 4 Characterization of cDNA Clones Encoding RB60

The BLASTX search using the EST sequences from clones listed in Table 5revealed similarity of the polypeptides encoded by the cDNAs to RB60from Chlamydomonas reinhardtii and to the putative protein disulfideisomerase-like protein from Arabidopsis thaliana resulting from the EUArabidopsis sequencing project (NCBI General Identifier Nos. 2708314 and4678297, respectively). Shown in Table 5 are the BLAST results forindividual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), or contigs assembled froman FIS and one or more ESTs (“Contig*”): TABLE 5 BLAST Results forSequences Encoding Polypeptides Homologousto RB60 BLAST pLog Score CloneStatus 2708314 4678297 fds.pk0022.c11 FIS 101.00 >254.00 Contig of:Contig* 95.00 157.00 cen3n.pk0155.e7 cs1.pk0100.a7 p0032.crcbb52rp0125.czabp07r cs1.pk0077.f10 FIS 47.15 83.52 sr1.pk0095.e9 FIS 34.3031.70 Contig of: Contig* 105.00 >254.00 scr1c.pk005.i17 sdp2c.pk038.e22sdp3c.pk021.a3 sfl1.pk0026.h1 sl2.pk0075.b10 wl1n.pk0027.f4 EST 58.3059.09 wre1n.pk0015.d10 FIS 59.00 92.00

The sequences from clones wl1n.pk0027.f4 and sr1l.pk0095.e9 also showedsimilarity to the predicted gene encoded by the contig of the rice ESTsD22477 and AU75323. The data in Table 6 represents a calculation of thepercent identity of the amino acid sequences set forth in SEQ ID NOs:8,10, 12, 14, 16, 18 and 20 and the Chlamydomonas reinhardtii andArabidopsis thaliana sequences (NCBI General Identifier Nos. 2708314 and4678297). TABLE 4 Percent Identity of Amino Acid Sequences Deduced Fromthe Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologousto RB60 Percent Identity to SEQ ID NO. 2708314 4678297 8 35.5 58.8 1032.0 47.9 12 39.5 66.4 14 28.3 25.9 16 34.8 57.0 18 27.3 25.9 20 35.453.4

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a balsam pear, two corn, two soybean andtwo wheat RB60. These sequences represent the first balsam pear, corn,soybean and wheat sequences encoding RB60.

Example 5 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embroys may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

1-15. (canceled)
 16. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having disulfide isomeraseactivity, wherein the amino acid sequence of the polypeptide and theamino acid sequence of SEQ ID NO:16 have at least 90% identity, or (b)the complement of the nucleotide sequence, wherein the complement andthe nucleotide sequence contain the same number of nucleotides and are100% complementary.
 17. The polynucleotide of claim 16 wherein the aminoacid sequence identity is at least 95%.
 18. The polynucleotide of claim16 wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:16.
 19. The polynucleotide of claim 16 wherein the polynucleotidecomprises the nucleotide sequence of SEQ ID NO:15.
 20. A chimeric genecomprising the polynucleotide of claim 16 operably linked to at leastone regulatory sequence.
 21. A cell comprising the polynucleotide ofclaim
 16. 22. The cell of claim 21, wherein the cell is selected fromthe group consisting of a yeast cell, a bacterial cell and a plant cell.23. A transgenic plant comprising the polynucleotide of claim
 16. 24. Avirus comprising the polynucleotide of claim
 16. 25. A method fortransforming a cell comprising introducing into a cell thepolynucleotide of claim
 16. 26. A method for producing a transgenicplant comprising (a) transforming a plant cell with the polynucleotideof claim 16 and (b) regenerating a plant from the transformed plantcell.
 27. A vector comprising the polynucleotide of claim
 16. 28. A seedcomprising the chimeric gene of claim
 20. 29. A method for isolating apolypeptide encoded by the polynucleotide of claim 16 comprisingisolating the polypeptide from a cell transformed with saidpolynucleotide.
 30. A composition comprising an isolated polynucleotidecomprising a nucleotide sequence encoding a first polypeptide of atleast 100 amino acids that has at least 85% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of a polypeptide of SEQ ID NOs:2, 4, 6, 8, 10, 12,14, 16, 18, and 20, or an isolated polynucleotide comprising thecomplement of the nucleotide sequence.